Bulletin of the American Physical Society
Annual Meeting of the APS Four Corners Section
Volume 60, Number 11
Friday–Saturday, October 16–17, 2015; Tempe, Arizona
Session I10: Biological Physics V: Advances in Spectroscopic Techniques |
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Chair: Carrie Moon, University of Denver Room: PSA106 |
Saturday, October 17, 2015 11:00AM - 11:24AM |
I10.00001: Biophysics of G-Protein-Coupled Receptor Activation in Membranes Invited Speaker: Michael Brown G-protein\textbf{-}coupled receptors (GPCRs) comprise almost 50{\%} of all pharmaceutical drug targets and afford enormous opportunities in biophysics. Here the visual protein rhodopsin is an important prototype and occurs naturally in lipid membranes. Photoactivation of rhodopsin entails 11-\textit{cis} to all-\textit{trans} isomerization of the bound retinal cofactor, yielding equilibrium between inactive Meta-I and active Meta-II states. We are employing solid-state nuclear magnetic resonance (NMR) spectroscopy as a powerful method to study rhodopsin activation in a membrane lipid environment [1]. For aligned membranes containing rhodopsin, the solid-state $^{\mathrm{2}}$H NMR lineshapes of the retinal cofactor determine its average conformation and orientation bound to the protein. Solid-state NMR data together with theoretical molecular dynamics (MD) simulations detect increased local mobility of retinal upon light activation [2]. The resulting changes in local dynamics of the cofactor initiate large-scale fluctuations of transmembrane helices that expose recognition sites for the signal-transducing G-protein. Moreover the lipids and water comprise the so-called "dark matter" of cellular membranes. Effects of membrane lipids on G-protein-coupled receptors (GPCRs) are revealed by UV-visible and FTIR spectroscopic studies of how they govern the conformational energetics of rhodopsin in visual signaling [3]. A new flexible surface model (FSM) describes how the curvature stress field of the membrane governs the energetics of active rhodopsin, due to the spontaneous monolayer curvature of the lipids [4]. The new biomembrane model challenges the standard fluid mosaic model. The FSM describes elastic coupling of membrane lipids to the conformational energetics of rhodopsin. Additional influences of osmotic pressure dictate that a large number of bulk water molecules are implicated in rhodopsin activation. An ensemble-mediated activation mechanism is proposed for rhodopsin in a natural membrane lipid environment, which includes a role of bulk water in the activation of rhodopsin-like GPCRs [4]. Ion channels, transporters, and membrane-bound peptides are all affected by elastic deformation of the bilayer, thus giving a new paradigm for membrane lipid-protein interactions in structural biophysics. [1] A. V. Struts et al. (2011)~\textit{PNAS}$~$\textbf{108}, 8263. [2] N. Leioatts et al. (2014)\textit{ Biochemistry} \textbf{53}, 376. [3] M. Mahalingam et al. (2008) \textit{PNAS} \textbf{105}, 17795. [4] M. F. Brown (2012) \textit{Biochemistry} \textbf{51}, 9782. [Preview Abstract] |
Saturday, October 17, 2015 11:24AM - 11:36AM |
I10.00002: Rhodopsin Activation in Membranes Studied by Solid-State NMR Spectroscopy Xiaolin Xu, Andrey Struts, Michael Brown Crystal structures of rhodopsin are available, yet the activation mechanism remains unknown. We introduced site-specific $^{2}$H labels into various methyl groups of the retinal cofactor and incorporated rhodopsin into membrane bilayers. Solid-state $^{2}$H NMR experiments were conducted for selectively deuterated retinal bound to rhodopsin in aligned samples in the active Meta-II state. The degree of alignment was tested by $^{31}$P NMR spectroscopy of the lipids. Solid-state $^{2}$H NMR lineshapes of the aligned samples were simulated by a static uniaxial distribution, which revealed the bond orientations of retinal methyl groups and mosaic spread [1]. Comparison with solid-state $^{2}$H NMR spectra predicted by X-ray results enabled the proposed structures for active Meta-II to be tested. Moreover, the dynamics of retinal were investigated by spin-lattice and quadrupolar-order relaxation measurements. Our generalized model-free method yields mean-squared amplitudes and correlation times of retinal bound to rhodopsin [2] as a basis for molecular dynamics (MD) simulations. Our broad aim is to establish how the local fluctuations of the ligand initiate the structural changes of rhodopsin to understand the activation mechanisms of GPCRs in general. [1] A.V. Struts et al. 2011, \textit{NSMB} \textbf{18}, 392. [2] X. Xu et al. 2014, \textit{eMagRes} \textbf{3}, 275. [Preview Abstract] |
Saturday, October 17, 2015 11:36AM - 11:48AM |
I10.00003: Biomolecules at the Interfaces of Nanostructured Materials Chengchen Guo, Gregory Holland, Jeffery Yarger To achieve the goal of applying biocompatible nanomaterials in developing biosensors and drug delivery systems relies on the fundamental understanding of the physical and chemical behavior of biomolecules at the interfaces of nanomaterials. Recently, researches have been making some progress in understanding the interaction between peptides/protein and various nanomaterials by using the simulation and modeling methods. However, experimental molecular level details focusing on the binding/interacting mechanism at nanoparticle (NP) surfaces are still lacking. A very first step towards the overall goal of understanding complicated biological systems is to understand how amino acids interact at the interfaces. Our group has been using a combination of nuclear magnetic resonance (NMR) techniques and optical spectroscopies to investigate these interactions and ultimately, to determine the structure of biomolecules at the surface of nanomaterials. We believe a better understanding of the molecular structure and dynamics of peptides and proteins at the interfaces of nanostructured materials will bring us closer to building devices that couple the unique properties of biomolecules with nanomaterials. Recent results from our research group on this topic will be discussed. [Preview Abstract] |
Saturday, October 17, 2015 11:48AM - 12:00PM |
I10.00004: Measuring the cytoskeletal properties of cell cultures using high-frequency ultrasound Caitlin Carter, Timothy Doyle High-frequency ultrasound (10-100 MHz) has been demonstrated to be sensitive to cell cytoskeletal changes. Cytoskeletal properties determine the biomechanical characteristics of cells and their role in many biomolecular processes. Examples include the aggressiveness and metastatic potential of breast cancer subtypes, T-cell activation during immune responses, and microtubule disintegration in Alzheimer's disease. The objectives of this work were to optimize the use of high-frequency ultrasound to subtype breast cancer cells and to acoustically measure cytoskeletal modifications. Pulse-echo measurements of 7 breast cancer cell lines of different molecular subtypes were acquired over a 2.5-year period using a 50-MHz transducer immersed in the growth media of monolayer cell cultures. Cell reflections were isolated from the interfering cell-culture plate reflections, spectrally analyzed using Gaussian curve fits, and spectrally classified using a heat map. The heat map displayed distinct patterns that differentiated the cell lines by molecular subtype. Cell cultures were also treated with colchicine and sphingosylphosphorylcholine to observe modulation of the microtubule and actin components. Cell waveforms and spectra displayed time-dependent changes due to chemical modification of the cytoskeleton. These results further verify and improve the noninvasive use of high-frequency ultrasound to differentiate breast cancer subtypes and to monitor cytoskeletal alterations in real time. [Preview Abstract] |
Saturday, October 17, 2015 12:00PM - 12:12PM |
I10.00005: High-Frequency Ultrasound for Evaluating Breast Cancer Surgical Margins: Micro-Tumor Detectability Study using Histology Mimicking Phantoms Nicole Cowan, Zachary Coffman, Robyn Omer, Benjamin Finch, Timothy Doyle The ability to differentiate between malignant and normal tissues in surgical margins during breast cancer surgery would reduce the risk of local recurrence and the need for subsequent surgeries. Clinical studies conducted in collaboration between Utah Valley University and the Huntsman Cancer Institute show that high-frequency (HF) ultrasound (20-80 MHz), and the parameters peak density and attenuation, are sensitive to breast tissue pathology. Pathology results from these clinical studies showed that many margin specimens contained micro-tumors measuring 1 mm in diameter or smaller. The objective of this study was to determine the sensitivity of HF ultrasound to these micro-tumors using histology mimicking phantoms. Phantoms were created from distilled water, agarose powder, 10X TBE stock solution, and microspheres to simulate tumors. Microspheres of 925?m diameter were embedded in the phantoms singularly and in clusters ranging from 3-12 microspheres. Pitch-catch measurements were acquired using large (low-resolution, 6.35mm diameter) and small (high-resolution, 1.5mm diameter) 50-MHz transducers, a HF pulser-receiver, a 1-GHz digital oscilloscope. Both large and small transducers were sensitive to single microspheres and microsphere clusters validating the clinical studies. [Preview Abstract] |
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